Callicarpa nudiflora extract as well as preparation method and application thereof

Abstract

The invention discloses a callicarpa nudiflora extract as well as a preparation method and application thereof, and relates to the technical field of traditional Chinese medicine extraction. The extract is prepared from cistanche glycoside D, rehmannia and cosmosiin. Compared with the prior art, the obtained callicarpa nudiflora extract has the advantages that the components are clear, the use safety is greatly improved, the synergistic anti-inflammatory effect is achieved, the anti-inflammatory effect is good, and the callicarpa nudiflora extract has a good application prospect.

Description

[0001] This invention is a divisional application of the Chinese patent with application number 202211280994.8, application date 2022.10.19, and invention name “A naked flower callicarpa extract, its preparation method and use”. Technical Field

[0002] The present invention relates to the technical field of traditional Chinese medicine extraction, and in particular to a Callicarpa nudiflora extract, a preparation method and use thereof. Background Art

[0003] Callicarpa nudiflora Hook.Et Arn. is a plant of the genus Callicarpa, Verbenaceae. Its stems, branches, and leaves are used medicinally, offering benefits such as dispersing blood stasis and swelling, combating bacteria and stopping bleeding, reducing inflammation and detoxifying, and dispelling wind and dampness. It is primarily used to treat suppurative inflammation, acute infectious hepatitis, respiratory and gastrointestinal bleeding, and traumatic bleeding. It can also be applied topically to treat burns, scalds, and hemorrhage from trauma. Research has shown that Callicarpa nudiflora contains flavonoids, terpenes, phenylpropanoids, and volatile oils, exhibiting pharmacological activities such as hemostasis, antithrombosis, anti-inflammatory, antibacterial, cytotoxic, and immune-enhancing properties.

[0004] Chinese patent application CN201610139149.7 discloses the use of a Callicarpa nudiflora extract in the preparation of a drug for treating gliomas, wherein the Callicarpa nudiflora extract is obtained by separating and extracting the aerial part of Callicarpa nudiflora, and the extraction steps include: (a) drying and crushing the aerial part of Callicarpa nudiflora, extracting with ethanol under hot reflux, combining the extracts, concentrating until there is no alcohol taste, and extracting with petroleum ether, ethyl acetate and water-saturated n-butanol in sequence to obtain a petroleum ether extract, an ethyl acetate extract and an n-butanol extract, respectively; (b) b) removing impurities from the n-butanol extract, first eluting with 10% ethanol for 6 column volumes, then eluting with 70% ethanol for 8 column volumes, collecting the 70% ethanol eluate, and concentrating under reduced pressure to obtain a 70% ethanol elution concentrate; (c) separating the 70% ethanol elution concentrate in step (b) using normal phase silica gel, eluting with a dichloromethane-methanol gradient, and collecting the eluate; then separating using octadecylsilane bonded reverse phase silica gel, isocratically eluting with a 70% volume percent methanol aqueous solution, collecting the eluate, and concentrating the eluate under reduced pressure to obtain the Callicarpa nudiflora extract.

[0005] Chinese patent application CN201510306705.0 discloses an extract of Callicarpa nudiflora with anti-inflammatory activity, containing 24-36% flavonoids, 0.8-3% phenylethanoid glycosides, and 15-27% diterpenoids. The extract is prepared by the following steps: (1) taking Callicarpa nudiflora medicinal material, grinding it into coarse powder, adding 90-95% ethanol, heating under reflux and extracting 1-3 times to obtain an extract, filtering the obtained filtrate and concentrating it into extract A; (2) uniformly dispersing the extract A obtained in step (1) in water, adding petroleum ether and ethyl acetate for extraction in turn, removing the solvent, and obtaining petroleum ether extraction effective fraction B and ethyl acetate extraction effective fraction C, respectively; (3) mixing effective fraction B and effective fraction C in a weight ratio of 1-3:1 to obtain the extract.

[0006] At present, the extract of Callicarpa nudiflora can only identify a few compounds, and cannot clarify all the components in the extract, resulting in uncertainty in the efficacy; in addition, specific extraction methods for specific pharmacologically active compounds in Callicarpa nudiflora are still relatively lacking and need to be improved. Summary of the Invention

[0007] The purpose of the present invention is to provide a Callicarpa nudiflora extract, a preparation method and use thereof, and to address the problems existing in the prior art, to provide a Callicarpa nudiflora extract with clear ingredients and good anti-inflammatory activity, and to provide a method for extracting specific ingredients from Callicarpa nudiflora.

[0008] Explanation of terms:

[0009] In the present invention, the mobile phase ratios are all volume ratios, and the purity percentages are all mass fractions.

[0010] In the present invention, numerical ranges include two endpoint values ​​and any real numbers therein. For example, "30-90%" includes but is not limited to 30%, 31%, 32%, 32.5%, 33.1%, 33.333%, 60%, 70%, 80%, 90%, etc.

[0011] In the present invention, the molecular structural formulas of the monomer compounds involved are as follows:

[0012] (6S,7R)-3-oxo-megastigma-4,8-dien-7-O-β-D-glucoside:

[0013]

[0014] Phoebenoside A:

[0015]

[0016] (6R,9R)-3-oxo-α-ionol-9-O-β-D-glucopyranoside:

[0017]

[0018] Blumenol C glucoside:

[0019]

[0020] Isovaleside:

[0021]

[0022] myricoside:

[0023]

[0024] Cistancheside D:

[0025]

[0026] Rehmannia glutinosa:

[0027]

[0028] calceolariosideB:

[0029]

[0030] 4,4'-dimethoxy-3'-hydroxy-7',9-diepoxylignan-3-O-β-D-glucopyranoside:

[0031] Luteolin-7-O-glucuronide:

[0032]

[0033] Luteolin-7-O-β-D-glucoside:

[0034]

[0035] Luteolin-4'-O-β-D-glucoside:

[0036]

[0037] Cosmoside:

[0038]

[0039] Luteolin-7-O-β-L-rhamnopyranosyl(1,2)-β-D-glucopyranoside:

[0040] Apiacetin-7-O-β-D-neohesperidin:

[0041]

[0042] 2-O-butyl-1-O-(2'-ethylhexyl)benzene-1,8-dicarboxylate:

[0043]

[0044] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0045] In one aspect, the present invention provides a Callicarpa nudiflora extract, characterized in that it comprises cistancheside D, rehmannia glutinosa and cosmosin.

[0046] Preferably, in the Callicarpa nudiflora extract, the mass ratio of cistancheside D, rehmannia glutinosa and cosmosin is 1-10:3-8:0.5-3.

[0047] In another aspect, the present invention provides a method for preparing the above-mentioned Callicarpa nudiflora extract, characterized in that it comprises the following steps:

[0048] S1, cold soaking of Callicarpa nudiflora leaves in water, followed by heating and decocting, combining the filtrates, and concentrating under reduced pressure to obtain extract 1, which was suspended in water, extracted with ethyl acetate, and the solvent was recovered under reduced pressure to obtain extract 2;

[0049] S2 and extract 2 were mixed with silica gel and crudely fractionated by silica gel column chromatography, with gradient elution using dichloromethane-methanol 600:1 to 1:1, to obtain 9 fractions Fr 1 to 9;

[0050] S3 and Fr 6 were separated on an MCI column and eluted with a methanol-water solvent system with a gradient of 30 to 90% methanol, and then combined to obtain Fr 6-1 to 6-5;

[0051] Among them, Fr 6-1 was subjected to silica gel column chromatography and gradient elution with dichloromethane-methanol 30:1 to 1:1 to obtain 6 components, Fr 6-1-1 to 6-1-6. Fr 6-1-3 was subjected to ODS column chromatography and isocratic elution with methanol-water 45-50:50-55, and preparative liquid phase was separated by 42-48% methanol and 40-45% methanol to obtain rehmannia glutinin; Fr 6-4 was separated by normal phase silica gel column chromatography and gradient elution with dichloromethane-methanol 80:1 to 1:1 to obtain 6 components, Fr 6-4-1 to 6-4-6; Fr 6-4-2 was separated by preparative liquid phase with 52-58% methanol to obtain cistancheside D; the three components with the largest extraction amount were selected and, in descending order of polarity, were Fr 6, Fr 4, and Fr 3.

[0052] S4 and Fr 4 were separated by reverse phase YMC column chromatography under pressure using a methanol-water solvent system gradient elution of 60-95% methanol. The three components with the largest extraction amount were selected in descending order of polarity, namely Fr 4-1, Fr 4-2, and Fr 4-3.

[0053] Among them, component Fr 4-3 was separated by Sephadex LH-20 column and methanol, and the two components with the largest extraction amount were selected, which were Fr 4-3-1 and Fr 4-3-3 in descending order of polarity; Fr 4-3-3 was separated by normal phase silica gel column chromatography, and four components Fr 4-3-3-1 to 4-3-3-5 were obtained by gradient elution with dichloromethane-methanol 30:1 to 1:1. Fr 4-3-3-1 was then subjected to preparative liquid chromatography with 42-48% methanol isocratic elution to obtain cosmosin.

[0054] In another aspect, the present invention provides a Callicarpa nudiflora extract comprising (6S,7R)-3-oxo-megastigma-4,8-dien-7-O-β-D-glucoside, phoebenoside A, (6R,9R)-3-oxo-α-ionol-9-O-β-D-glucopyranoside, blumenol C glucoside, isovanoscoside, myricoside, cistanchoside D, rutin, calceolarioside B, 4,4'-dimethoxy-3'-hydroxy-7',9-diepoxylignan-3-O-β-D-glucopyranoside, luteolin-7-O-glucuronide, luteolin-7-O-β-D-glucoside, luteolin-4'-O-β-D-glucoside, cosmosin, luteolin-7-O-β-L-rhamnopyranosyl(1,2)-β-D-glucopyranoside, apiol-7-O-β-D-neohesperidin, and 2-O-butyl-1-O-(2'-ethylhexyl)benzene-1,8-dicarboxylate.

[0055] Preferably, in the Callicarpa nudiflora extract, (6S,7R)-3-oxo-megastigma-4,8-dien-7-O-β-D-glucoside, phoebenoside A, (6R,9R)-3-oxo-α-ionol-9-O-β-D-glucopyranoside, blumenol C glucoside, isocarpus glycoside, myricoside, cistancheside D, rutin, calceolarioside B, 4,4'-dimethoxy-3'-hydroxy-7',9-diepoxylignan-3-O-β-D-glucopyranoside, luteolin-7-O-glucuronide, luteolin-7-O-β-D-glucoside, luteolin-4'-O-β-D-glucoside, cosmosin, luteolin-7-O-β-L-rhamnopyranosyl(1,2)-β-D-glucopyranoside The mass ratio of ranoside, apigenin-7-O-β-D-neohesperidin and 2-O-butyl-1-O-(2'-ethylhexyl)benzene-1,8-dicarboxylate is 1-5:5-15:1-5:2-6:0.5-5:10-15:1-10:0.1-1.5:1-5:3-8:0.5-4:3-8:0.5-3:0.5-3:1-3:1-5:0.5-4; more preferably 3.2:8:2.2:3.8:1.7:12:4.2:0.8:2.2:5.4:1.2:5.4:1.6:1.1:1.8:2.3:1.3.

[0056] In another aspect, the present invention provides a method for preparing the above-mentioned Callicarpa nudiflora extract, comprising the following steps:

[0057] S1, cold soaking of Callicarpa nudiflora leaves in water, followed by heating and decocting, combining the filtrates, and concentrating under reduced pressure to obtain extract 1, which was suspended in water, extracted with ethyl acetate, and the solvent was recovered under reduced pressure to obtain extract 2;

[0058] S2 and extract 2 were mixed with silica gel and crudely separated by silica gel column chromatography. Gradient elution was performed with dichloromethane-methanol 600:1 to 1:1. The three components with the largest extraction amount were selected, and the order of polarity from high to low was Fr 6, Fr 4, and Fr 3;

[0059] S3 and Fr 6 were separated on an MCI column and eluted with a methanol-water solvent system with a gradient of 30 to 90% methanol. The two components with the largest extraction amounts were selected, and in descending order of polarity, they were Fr 6-1-4 and Fr 6-1-3;

[0060] Among them, Fr 6-1 was subjected to silica gel column chromatography with a gradient elution of dichloromethane-methanol 30:1 to 1:1, and the two components with the largest extraction amount were selected, in descending order of polarity, as Fr 6-1-4 and Fr 6-1-3;

[0061] Fr 6-1-3 was subjected to ODS column chromatography with isocratic elution of methanol-water at a ratio of 45-50:50-55, and isocratic separation using 42-48% methanol and 40-45% methanol, respectively, to give compounds 8 and 11; Fr 6-1-4 was further separated by preparative liquid chromatography using 43-46% methanol and 45-50% methanol, respectively, to give compounds 15 and 16;

[0062] Fr 6-4 was separated by normal phase silica gel column chromatography, and the two components with the largest extraction amount were selected in descending order of polarity, namely Fr 6-4-2 and Fr 6-4-1;

[0063] Fr 6-4-1 was separated by preparative liquid phase with 50-55% methanol to obtain compound 1; Fr 6-4-2 was separated by preparative liquid phase with 52-58% methanol to obtain compounds 4 and 7;

[0064] S4 and Fr 4 were separated by reverse phase YMC column chromatography under pressure using a methanol-water solvent system gradient elution of 60-95% methanol. The three components with the largest extraction amount were selected in descending order of polarity, namely Fr 4-1, Fr 4-2, and Fr 4-3.

[0065] Among them, Fr 4-1 was separated by normal phase silica gel column chromatography, and dichloromethane-methanol 20:1 to 1:1 gradient elution was used to obtain compounds 2 and 6; component Fr4-2 was separated on a silica gel column, and dichloromethane-methanol 50:1 to 5:1 gradient elution was used; and then the preparative liquid phase was passed through 52-58% methanol to obtain compounds 3 and 10;

[0066] Component Fr 4-3 was separated by Sephadex LH-20 column and methanol, and the two components with the largest extraction amount were selected, which were Fr 4-3-1 and Fr 4-3-3 in descending order of polarity;

[0067] Fr 4-3-1 was separated by normal phase silica gel column chromatography with a gradient elution of dichloromethane-methanol from 20:1 to 5:1 to obtain compounds 9 and 12; the elution ratios were dichloromethane:methanol at 10:1 and 7:1; Fr 4-3-3 was separated by normal phase silica gel column chromatography with a gradient elution of dichloromethane-methanol from 30:1 to 1:1, and the two components with the largest extraction amount were selected, in descending order of polarity, as Fr 4-3-3-3 and Fr 4-3-3-1.

[0068] Fr 4-3-3-1 was further purified by preparative liquid phase with 42-48% methanol isocratic elution to give compounds 13 and 14; Fr 4-3-3-3 was further purified by preparative liquid phase with 42-50% methanol to give compound 5;

[0069] S5 and Fr 3 were separated on an MCI column, and then eluted with a methanol-water solvent system of 40-95% methanol by gradient elution, and then separated by preparative liquid chromatography with 45-55% methanol to obtain compound 17.

[0070] Among them, compounds 1-17 are (6S,7R)-3-oxo-megastigma-4,8-dien-7-O-β-D-glucoside, phoebenoside A, (6R,9R)-3-oxo-α-ionol-9-O-β-D-glucopyranoside, blumenol C, respectively. glucoside, isocarpus glycoside, myricoside, cistancheside D, rutin, calceolarioside B, 4,4'-dimethoxy-3'-hydroxy-7',9-diepoxylignan-3-O-β-D-glucopyranoside, luteolin-7-O-glucuronide, luteolin-7-O-β-D-glucoside, luteolin-4'-O-β-D-glucoside, cosmosin, luteolin-7-O-β-L-rhamnopyranosyl(1,2)-β-D-glucopyranoside, apiol-7-O-β-D-neohesperidin, 2-O-butyl-1-O-(2'-ethylhexyl)benzene-1,8-dicarboxylate.

[0071] Preferably, in step S1, the time for cold soaking in water is 6-18 hours, the number of heating and decocting is 2-5 times, each time for 1-5 hours, and the number of ethyl acetate extraction is 1-5 times.

[0072] Preferably, in step S2, the volume ratio of dichloromethane to methanol is 400-600:1, 200-400:1, 100-300:1, 50-150:1, 50-100:1, 15-40:1, 10-20:1, 5-15:1, or 1-8:1. As a specific embodiment of the present invention, gradient elution is performed according to the following volume ratios: 500:1, 300:1, 200:1, 100:1, 70:1, 20:1, 15:1, 10:1, or 5:1.

[0073] Preferably, in step S3, the methanol-water solvent system is 30-60% methanol, 50-70% methanol, 60-80% methanol, 70-90% methanol, and 85-90% methanol for elution. As a specific embodiment of the present invention, gradient elution is performed according to the following volume ratio: 50% methanol, 60% methanol, 70% methanol, 80% methanol, and 90% methanol.

[0074] The volume ratios of dichloromethane to methanol used for eluting Fr 6-1 were 10-30:1, 10-20:1, 10-15:1, 5-12:1, 3-10:1, and 1-8:1. As a specific embodiment of the present invention, gradient elution was performed according to the following volume ratios: 20:1, 15:1, 12:1, 10:1, 8:1, and 6:1.

[0075] The volume ratios of dichloromethane to methanol used for elution of Fr 6-4 were 60-80:1, 20-40:1, 15-30:1, 10-20:1, 5-15:1, and 1-8:1. As a specific embodiment of the present invention, gradient elution was performed according to the following volume ratios: 70:1, 30:1, 20:1, 15:1, 10:1, and 5:1.

[0076] Preferably, in step S4, the methanol-water solvent system is 60-70% methanol, 70-80% methanol, 80-90% methanol, 90-95% methanol. As a specific embodiment of the present invention, gradient elution is performed according to the following volume ratios: 65% methanol, 75% methanol, 85% methanol, 95% methanol.

[0077] The volume ratio of dichloromethane to methanol used for eluting Fr 4-1 is 10-20: 1, 1-10: 1. As a specific embodiment of the present invention, gradient elution is performed according to the following volume ratios: 10: 1, 8: 1.

[0078] The volume ratios of dichloromethane to methanol used for eluting Fr 4-2 are 25-50:1, 15-25:1, 10-20:1, 8-15:1, and 5-10:1. As a specific embodiment of the present invention, gradient elution is performed according to the following volume ratios: 30:1, 20:1, 15:1, 10:1, and 5:1.

[0079] The volume ratios of dichloromethane to methanol used for eluting Fr 4-3-1 were 10-20:1 and 5-10:1. As a specific embodiment of the present invention, gradient elution was performed according to the following volume ratios: 10:1 and 7:1.

[0080] The volume ratios of dichloromethane to methanol used for eluting Fr 4-3-3 were 15-30:1, 10-20:1, 5-15:1, and 1-10:1. As a specific embodiment of the present invention, gradient elution was performed according to the following volume ratios: 20:1, 15:1, 10:1, and 5:1.

[0081] Preferably, in step S5, the methanol-water solvent system is 40-60% methanol, 50-70% methanol, 60-80% methanol, 70-85%, 80-95% methanol. As a specific embodiment of the present invention, gradient elution is performed according to the following volume ratios: 50% methanol, 60% methanol, 70% methanol, 80% methanol, 90% methanol.

[0082] In another aspect, the present invention provides a medicine of the above-mentioned Callicarpa nudiflora extract.

[0083] In another aspect, the present invention provides a method for extracting (6S,7R)-3-oxo-megastigma-4,8-dien-7-O-β-D-glucoside from Callicarpa nudiflora, comprising the following steps:

[0084] S1, cold soaking of Callicarpa nudiflora leaves in water, followed by heating and decocting, combining the filtrates, and concentrating under reduced pressure to obtain extract 1, which was suspended in water, extracted with ethyl acetate, and the solvent was recovered under reduced pressure to obtain extract 2;

[0085] S2 and extract 2 were mixed with silica gel and crudely fractionated by silica gel column chromatography, with gradient elution using dichloromethane-methanol 600:1 to 1:1, to obtain 9 fractions Fr 1 to 9;

[0086] S3 and Fr 6 were separated on an MCI column and eluted with a methanol-water solvent system with a gradient of 30-90% methanol, and Fr 6-1 to 6-5 were obtained by combination;

[0087] Among them, Fr 6-1 was purified by silica gel column chromatography with a gradient elution of dichloromethane-methanol 30:1 to 1:1 to obtain 6 components, Fr 6-1-1 to 6-1-6; Fr 6-1-4 was further purified by preparative liquid phase to obtain compounds 15 and 16;

[0088] Fr 6-4 was separated by normal phase silica gel column chromatography and eluted with a gradient of dichloromethane-methanol 80:1 to 1:1 to obtain 6 components, Fr 6-4-1 to 6-4-4. Fr 6-4-1 was purified by preparative liquid phase to obtain (6S,7R)-3-oxo-megastigma-4,8-dien-7-O-β-D-glucoside.

[0089] In another aspect, the present invention provides a method for extracting phoebenoside A from Callicarpa nudiflora, comprising the following steps:

[0090] S1, cold soaking of Callicarpa nudiflora leaves in water, followed by heating and decocting, combining the filtrates, and concentrating under reduced pressure to obtain extract 1, which was suspended in water, extracted with ethyl acetate, and the solvent was recovered under reduced pressure to obtain extract 2;

[0091] S2 and extract 2 were mixed with silica gel and crudely fractionated by silica gel column chromatography, with gradient elution using dichloromethane-methanol 600:1 to 1:1, to obtain 9 fractions Fr 1 to 9;

[0092] S3 and Fr 4 were separated by pressure chromatography on a reverse-phase YMC column using a gradient elution system of methanol-water with a ratio of 60-95% methanol to obtain four components, Fr 4-1 to 4-4. Among them, Fr 4-1 was separated by normal-phase silica gel column chromatography using a gradient elution system of dichloromethane-methanol (20:1 to 1:1). The dichloromethane:methanol (7:1) fractions were collected to obtain phoebenoside A.

[0093] In another aspect, the present invention provides a method for extracting (6R,9R)-3-oxo-α-ionol-9-O-β-D-glucopyranoside from Callicarpa nudiflora, comprising the following steps:

[0094] S1, cold soaking of Callicarpa nudiflora leaves in water, followed by heating and decocting, combining the filtrates, and concentrating under reduced pressure to obtain extract 1, which was suspended in water, extracted with ethyl acetate, and the solvent was recovered under reduced pressure to obtain extract 2;

[0095] S2 and extract 2 were mixed with silica gel and crudely fractionated by silica gel column chromatography, with gradient elution using dichloromethane-methanol 600:1 to 1:1, to obtain 9 fractions Fr 1 to 9;

[0096] S3 and Fr 4 were separated by reverse phase YMC column chromatography under pressure and eluted with a methanol-water solvent system of 60-95% methanol to obtain four components Fr 4-1 to 4-4;

[0097] Among them, component Fr4-2 was separated on a silica gel column, and after gradient elution with dichloromethane-methanol 50:1 to 5:1, the 10:1 elution segment was collected and then purified by high performance liquid phase to obtain (6R,9R)-3-oxo-α-ionol-9-O-β-D-glucopyranoside.

[0098] In another aspect, the present invention provides a method for extracting blumenol C glucoside from Callicarpa nudiflora, comprising the following steps:

[0099] S1, cold soaking of Callicarpa nudiflora leaves in water, followed by heating and decocting, combining the filtrates, and concentrating under reduced pressure to obtain extract 1, which was suspended in water, extracted with ethyl acetate, and the solvent was recovered under reduced pressure to obtain extract 2;

[0100] S2 and extract 2 were mixed with silica gel and crudely fractionated by silica gel column chromatography, with gradient elution using dichloromethane-methanol 600:1 to 1:1, to obtain 9 fractions Fr 1 to 9;

[0101] S3 and Fr 6 were separated on an MCI column and eluted with a methanol-water solvent system with a gradient of 30-90% methanol to obtain Fr 6-1 to 6-5;

[0102] Among them, Fr 6-4 was separated by normal phase silica gel column chromatography and gradient elution with dichloromethane-methanol 80:1 to 1:1 to obtain 6 components, Fr6-4-1 to 6-4-4. Fr 6-4-2 was purified by high performance liquid chromatography to obtain blumenol Cglucoside.

[0103] In another aspect, the present invention provides a method for extracting calceolarioside B from Callicarpa nudiflora, comprising the following steps:

[0104] S1, cold soaking of Callicarpa nudiflora leaves in water, followed by heating and decocting, combining the filtrates, and concentrating under reduced pressure to obtain extract 1, which was suspended in water, extracted with ethyl acetate, and the solvent was recovered under reduced pressure to obtain extract 2;

[0105] S2 and extract 2 were mixed with silica gel and crudely fractionated by silica gel column chromatography, with gradient elution using dichloromethane-methanol 600:1 to 1:1, to obtain 9 fractions Fr 1 to 9;

[0106] S3 and Fr 4 were separated by reverse phase YMC column chromatography under pressure and eluted with a methanol-water solvent system of 60-95% methanol to obtain four components Fr 4-1 to 4-4;

[0107] Among them, component Fr 4-3 was separated by Sephadex LH-20 column and methanol, and combined to obtain 5 components, Fr 4-3-1 to 4-3-5. Fr 4-3-1 was separated by normal phase silica gel column chromatography and eluted with dichloromethane-methanol 20:1 to 5:1 gradient to obtain calceolarioside B.

[0108] In another aspect, the present invention provides a method for extracting 4,4'-dimethoxy-3'-hydroxy-7',9-diepoxylignan-3-O-β-D-glucopyranoside from Callicarpa nudiflora, comprising the following steps:

[0109] S1, cold soaking of Callicarpa nudiflora leaves in water, followed by heating and decocting, combining the filtrates, and concentrating under reduced pressure to obtain extract 1, which was suspended in water, extracted with ethyl acetate, and the solvent was recovered under reduced pressure to obtain extract 2;

[0110] S2 and extract 2 were mixed with silica gel and crudely fractionated by silica gel column chromatography, with gradient elution using dichloromethane-methanol 600:1 to 1:1, to obtain 9 fractions Fr 1 to 9;

[0111] S3 and Fr 4 were separated by reverse phase YMC column chromatography under pressure and eluted with a methanol-water solvent system of 60-95% methanol to obtain four components Fr 4-1 to 4-4;

[0112] Among them, component Fr4-2 was separated on a silica gel column, eluted with a gradient of dichloromethane-methanol 50:1 to 5:1, and then purified by preparative liquid phase to obtain compound 4,4'-dimethoxy-3'-hydroxy-7',9-diepoxylignan-3-O-β-D-glucopyranoside.

[0113] In another aspect, the present invention provides a method for extracting luteolin-7-O-glucuronide from Callicarpa nudiflora, comprising the following steps:

[0114] S1, cold soaking of Callicarpa nudiflora leaves in water, followed by heating and decocting, combining the filtrates, and concentrating under reduced pressure to obtain extract 1, which was suspended in water, extracted with ethyl acetate, and the solvent was recovered under reduced pressure to obtain extract 2;

[0115] S2 and extract 2 were mixed with silica gel and crudely fractionated by silica gel column chromatography, with gradient elution using dichloromethane-methanol 600:1 to 1:1, to obtain 9 fractions Fr 1 to 9;

[0116] S3 and Fr 6 were separated on an MCI column and eluted with a methanol-water solvent system with a gradient of 30-90% methanol, and Fr 6-1 to 6-5 were obtained by combination;

[0117] Among them, Fr 6-1 was subjected to silica gel column chromatography with a dichloromethane-methanol gradient elution to obtain 6 components, Fr 6-1-1 to 6-1-6; Fr 6-1-3 was subjected to ODS column chromatography with a 45-50:50-55 methanol-water isocratic elution and preparative liquid phase separation to obtain luteolin-7-O-glucuronide.

[0118] In another aspect, the present invention provides a method for extracting 2-O-butyl-1-O-(2′-ethylhexyl)benzene-1,8-dicarboxylate from Callicarpa nudiflora, comprising the following steps:

[0119] S1, cold soaking of Callicarpa nudiflora leaves in water, followed by heating and decocting, combining the filtrates, and concentrating under reduced pressure to obtain extract 1, which was suspended in water, extracted with ethyl acetate, and the solvent was recovered under reduced pressure to obtain extract 2;

[0120] S2 and extract 2 were mixed with silica gel and crudely fractionated by silica gel column chromatography, with gradient elution using dichloromethane-methanol 600:1 to 1:1, to obtain 9 fractions Fr 1 to 9;

[0121] S3 and Fr 3 were separated on an MCI column and eluted with a methanol-water solvent system with a gradient of 40-95% methanol, and then separated by preparative liquid chromatography with 45-55% methanol to obtain 2-O-butyl-1-O-(2′-ethylhexyl)benzene-1,8-dicarboxylate.

[0122] On the other hand, the present invention provides the use of the above-mentioned Callicarpa nudiflora extract, or the Callicarpa nudiflora extract prepared by the above-mentioned preparation method, or the extract prepared by the above-mentioned extraction method in the preparation of therapeutic anti-inflammatory drugs.

[0123] Finally, the present invention provides the use of Cistancheside D in the preparation of anti-inflammatory drugs.

[0124] In the present invention, the use of the Cistancheside D and Callicarpa nudiflora extract includes the use of Cistancheside D and Callicarpa nudiflora extract as the sole active ingredient, or the use of Cistancheside D and Callicarpa nudiflora extract in combination with at least one other anti-inflammatory active ingredient.

[0125] The active ingredient can be administered orally in solid dosage forms such as capsules, tablets, lozenges, troches, granules, and powders, or in liquid dosage forms such as elixirs, syrups, emulsions, dispersions, and suspensions. The active ingredient can also be administered parenterally in sterile liquid dosage forms such as dispersions, suspensions, or solutions. Other dosage forms that can be used to administer the active ingredient include ointments, creams, drops, transdermal patches, or powders for topical administration; ophthalmic solutions or suspensions, i.e., eye drops, for administration to the eye; sprays or powder compositions for inhalation or intranasal administration, or creams, ointments, sprays, or suppositories for rectal or vaginal administration. Gelatin capsules contain the active ingredient and a powdered carrier such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Similar diluents can be used to prepare compressed tablets. Both tablets and capsules can be formulated as sustained-release products to provide sustained release of the drug over several hours. Compressed tablets can be coated with sugar or film to cover any unpleasant taste and protect the tablet from air, or can be enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can include coloring agents and flavoring agents to increase patient acceptance. Generally speaking, water, suitable oil, saline, dextrose (glucose) aqueous solution and related sugar solutions and glycols such as propylene glycol or polyethylene glycol are suitable carriers of parenteral solutions. The solution for parenteral administration preferably includes a water-soluble salt of the active ingredient, a suitable stabilizer and the buffer substance used as needed. Antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid, alone or in combination, are suitable stabilizers. Citric acid and its salts and sodium EDTA can also be used. In addition, parenteral solutions can also include preservatives, such as benzalkonium chloride, methylparaben or propylparaben and chlorobutanol. For inhalation administration, the compound of the present invention can be easily delivered in a spray form from a pressurized package or a sprayer. This compound can also be sent in the powder form that prepares, and this powder composition can be sucked with the help of blowing into powder inhaler device.Preferred delivery system for sucking is metered dose inhalation (MDI) aerosol, and it can be mixed with suspension or the solution of active component in suitable propellant, and described propellant is fluorocarbon or hydrocarbon for example.For eye administration, ophthalmic preparation can prepare with the solution or the suspension of the suitable weight percentage of active component in suitable ophthalmic carrier, thereby keeps compound and eye surface contact enough time so that compound is penetrated into cornea and the inner area of ​​eyes.

[0126] The beneficial effects of the present invention are:

[0127] To address the problems of the existing technology, a Callicarpa nudiflora extract with a well-defined composition and excellent anti-inflammatory activity and a preparation method thereof are provided, as well as a method for extracting specific components from Callicarpa nudiflora. Compared with existing technologies, the obtained Callicarpa nudiflora extract has a well-defined composition and greatly improved safety. It also has excellent anti-inflammatory effects and has promising application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0128] Figure 1 It is the extraction process flow chart of the present invention. DETAILED DESCRIPTION

[0129] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further illustrated below in conjunction with specific examples, but the following examples are only preferred embodiments of the present invention, not all. Based on the examples in the implementation manner, other embodiments obtained by those skilled in the art without making creative work are all within the scope of protection of the present invention. In the following examples, unless otherwise specified, the operating methods used are all conventional operating methods, the equipment used are all conventional equipment, and the equipment and materials used in each embodiment are all the same.

[0130] In the following examples, the medicinal material of Callicarpa nudiflora was purchased from Hengshengyuan Planting Professional Cooperative in Baisha, Hainan Province, and was identified as Callicarpa nudiflora by Professor Fan Cuisheng of Jiangxi University of Traditional Chinese Medicine. The voucher specimen (No. Y202101G) was stored in the Jiangzhong Medicine Valley Research Center.

[0131] Bruker Avance-ⅢHD 600 MHz nuclear magnetic resonance spectrometer (Bruker, Germany); Triple TOF5600 high-resolution time-of-flight mass spectrometer (AB Sciex, USA); ThermoUItiMate 3000 high-performance liquid chromatography (Thermo Fisher Scientific, USA); Elite P3500 semi-preparative liquid chromatograph (Dalian Elite Analytical Instrument Co., Ltd.); YMC-Triart C18 semi-preparative chromatographic column (10 mm × 250 mm, 5 μm, YMC, Japan); MCI-gel (75-150 μm, Mitsubishi Chemical, Japan); Sephadex LH-20 dextran gel (GE Healthcare, USA) Healthcare Company); column chromatography silica gel (100-200 mesh, 200-300 mesh, Qingdao Ocean Chemical Plant); MS204S / 01 analytical balance (Mettler Toledo, Switzerland); Milli-Q ultrapure water machine (Millipore, USA); lipopolysaccharide and dexamethasone (Sigma, USA); phosphate buffer and DMEM high-glucose medium (Solarbio, China); fetal bovine serum (Gibco, USA); NO and CCK8 kits (Shanghai Biyuntian Biotechnology Co., Ltd.); all reagents used were of chromatographic or analytical grade.

[0132] Example 1

[0133] 3.0 kg of dried leaves of Callicarpa nudiflora were added to water and cold soaked for 12 h, then heated and decocted three times, each time for 3 h. The filtrate was filtered and combined while hot, and concentrated under reduced pressure to an extract. The extract was suspended and dispersed with an appropriate amount of water, added with an appropriate amount of ethyl acetate, extracted three times, and the solvent was recovered under reduced pressure to obtain 314 g of extract. After mixing with silica gel, the sample was crudely fractionated by silica gel column chromatography, and gradient elution was performed with dichloromethane:methanol 500:1, 300:1, 200:1, 100:1, 70:1, 20:1, 15:1, 10:1, and 5:1. Thin layer chromatography was used to preliminarily determine the selection criteria based on the height of the fraction developed on the silica gel plate. The fractions with similar development heights were merged, and finally 9 fractions Fr 1 to 9 were obtained; the developing solvent selected for the Fr1 part was dichloromethane-methanol 30:1, the developing solvent selected for the Fr2 part was dichloromethane-methanol 25:1, the developing solvent selected for the Fr3 part was dichloromethane-methanol 22:1, the developing solvent selected for the Fr4 part was dichloromethane-methanol 20:1, the developing solvent selected for the Fr5 part was dichloromethane-methanol 15:1, the developing solvent selected for the Fr6 part was dichloromethane-methanol 12:1, and the developing solvent selected for the Fr7 part was dichloromethane-methanol 13:1. The developing solvent selected for part 7 was dichloromethane-methanol 10:1, the developing solvent selected for part Fr 8 was dichloromethane-methanol 8:1, and the developing solvent selected for part Fr 9 was dichloromethane-methanol 5:1;

[0134] The three components with the largest extraction amounts were selected, and in descending order of polarity, they were Fr 6, Fr 4, and Fr 3.

[0135] Fr 6 (30.14 g) was separated on an MCI column and gradient eluted with a methanol-water solvent system (50% methanol, 60% methanol, 70% methanol, 80% methanol, 90% methanol). Thin-layer chromatography was used as the selection criterion for the development height of the fractions on the silica gel plate. Fractions with similar development heights were combined to obtain Fr6-1 to 6-5. The developing solvent selected for the Fr 6-1 portion was dichloromethane-methanol 12:1, the developing solvent selected for the Fr 6-2 portion was dichloromethane-methanol 15:1, the developing solvent selected for the Fr 6-3 portion was dichloromethane-methanol 12:1, the developing solvent selected for the Fr 6-4 portion was dichloromethane-methanol 10:1, and the developing solvent selected for the Fr 6-5 portion was dichloromethane-methanol 8:1.

[0136] The two components with the largest extraction amount were selected, and their polarity was ranked from high to low, namely Fr 6-1 and Fr 6-4.

[0137] Among them, Fr 6-1 (8.11 g) was subjected to silica gel column chromatography and gradient elution with dichloromethane-methanol to obtain 6 components, Fr6-1-1 to 6-1-6; among them, dichloromethane:methanol 20:1, 15:1, 12:1, 10:1, 8:1, 6:1, and thin layer chromatography was preliminarily judged to be the selection standard based on the height of the fraction developed on the silica gel plate. The fractions with similar development heights were combined to obtain 6 components, Fr 6-1-1 to 6-1-6. The developing solvent selected for Fr 6-1-1 was dichloromethane-methanol 12:1, the developing solvent selected for Fr 6-1-2 was dichloromethane-methanol 12:1, the developing solvent selected for Fr 6-1-3 was dichloromethane-methanol 12:1, the developing solvent selected for Fr 6-1-4 was dichloromethane-methanol 10:1, and the developing solvent selected for Fr The developing solvent selected for section 6-1-5 was dichloromethane-methanol 10:1; the developing solvent selected for section Fr 6-1-6 was dichloromethane-methanol 10:1;

[0138] The two components with the largest extraction amount were selected, and their order of polarity from high to low was Fr 6-1-4 and Fr 6-1-3.

[0139] Fr 6-1-3 (1.04 g) was repeatedly separated and purified by ODS column chromatography, eluting with methanol-water (48:52), and preparative liquid chromatography with 43% methanol to give compounds 8 (0.8 mg) and 11 (1.2 mg). Fr 6-1-4 (0.95 g) was further purified by semi-preparative HPLC with 54% methanol to give compounds 15 (1.8 mg) and 16 (2.3 mg).

[0140] Fr 6-4 (5.81 g) was separated by normal phase silica gel column chromatography and eluted with dichloromethane-methanol gradient to obtain 6 components, Fr6-1-1 to 6-1-6; the dichloromethane:methanol ratio was 20:1, 15:1, 12:1, 10:1, 8:1, and 6:1, respectively. Thin-layer chromatography was used as the preliminary judgment standard for the selection of fractions with similar development heights. The developing solvent selected for Fr6-4-1 was dichloromethane-methanol 10:1, the developing solvent selected for Fr 6-4-2 was dichloromethane-methanol 10:1, the developing solvent selected for Fr 6-4-3 was dichloromethane-methanol 10:1, the developing solvent selected for Fr 6-4-4 was dichloromethane-methanol 8:1, the developing solvent selected for Fr6-4-5 was dichloromethane-methanol 8:1; and the developing solvent selected for Fr 6-4-6 was dichloromethane-methanol 8:1.

[0141] The two components with the largest extraction amount were selected, and their order of polarity from high to low was Fr 6-4-2 and Fr 6-4-1.

[0142] Fr 6-4-1 was purified by semi-preparative HPLC with 49% methanol to give compound 1 (3.2 mg). Fr 6-4-2 was purified by semi-preparative HPLC with 44% methanol to give compounds 4 (3.8 mg) and 7 (4.2 mg).

[0143] Fr 4 (86.82 g) was separated by pressure chromatography on a reverse-phase YMC column, and then gradient eluted with methanol-water (95% methanol, 85% methanol, 75% methanol, 65% methanol). Thin-layer chromatography was performed to preliminarily determine that the height of the fractions developed on the silica gel plate was used as the selection criterion, and fractions with similar development heights were combined to obtain four components Fr 4-1 to 4-4. The developing solvent selected for the Fr 4-1 fraction was dichloromethane-methanol 12:1, the developing solvent selected for the Fr 4-2 fraction was dichloromethane-methanol 12:1, the developing solvent selected for the Fr 4-3 fraction was dichloromethane-methanol 10:1, and the developing solvent selected for the Fr 4-4 fraction was dichloromethane-methanol 10:1.

[0144] The three components with the largest extraction amounts were selected, and in descending order of polarity, they were Fr 4-1, Fr 4-2, and Fr 4-3.

[0145] Fr 4-1 (12.37 g) was separated by normal phase silica gel column chromatography using dichloromethane:methanol ratios of 10:1 and 8:1 to obtain compounds 2 (8 mg) and 6 (12 mg). Fraction Fr 4-2 (9.12 g) was separated on a silica gel column using a dichloromethane-methanol gradient elution ratio of 30:1, 20:1, 15:1, 10:1, and 5:1; it was then purified by semi-preparative HPLC with 51% methanol to obtain compounds 3 (2.2 mg) and 10 (5.4 mg).

[0146] Component Fr 4-3 (6.34 mg) was separated on a Sephadex LH-20 column with dichloromethane-methanol (1:1). Thin-layer chromatography was used to preliminarily determine the fractions, and the fractions with similar development heights on the silica gel plate were combined to obtain five fractions, Fr 4-3-1 to 4-3-5. The developing solvent selected for Fr 4-3-1 was dichloromethane-methanol 10:1, the developing solvent selected for Fr 4-3-2 was dichloromethane-methanol 10:1, the developing solvent selected for Fr 4-3-3 was dichloromethane-methanol 10:1, the developing solvent selected for Fr 4-3-4 was dichloromethane-methanol 8:1, and the developing solvent selected for Fr 4-3-5 was dichloromethane-methanol 8:1.

[0147] The two components with the largest extraction amount were selected, and their order of polarity from high to low was Fr 4-3-1 and Fr 4-3-3.

[0148] Fr 4-3-1 (1.34 g) was separated by normal phase silica gel column chromatography with a gradient elution of dichloromethane-methanol (20:1 to 5:1) in an elution ratio of dichloromethane:methanol of 30:1, 20:1, 15:1, 10:1, and 5:1; compounds 9 (2.2 mg) (10:1 elution fraction) and 12 (5.4 mg) (5:1 elution fraction) were obtained.

[0149] Fr 4-3-3 (2.54 g) was separated by normal phase silica gel column chromatography with a gradient elution of dichloromethane-methanol (20:1 to 5:1) at elution ratios of dichloromethane:methanol of 30:1, 20:1, 15:1, 10:1, and 5:1. Thin layer chromatography analysis revealed that fractions with similar development heights on the silica gel plate were selected as the selection criteria, and fractions with similar development heights were combined to obtain four components, Fr 4-3-3-1 to 4-3-3-4. The developing solvent used for Fr 4-3-3-1 was dichloromethane-methanol (10:1), the developing solvent used for Fr 4-3-3-2 was dichloromethane-methanol (10:1), the developing solvent used for Fr4-3-3-3 was dichloromethane-methanol (10:1), and the developing solvent used for Fr4-3-3-4 was dichloromethane-methanol (8:1).

[0150] The two components with the largest extraction amount were selected, and their order of polarity from high to low was Fr 4-3-3-3 and Fr4-3-3-1.

[0151] Fr 4-3-3-1 was further purified by semi-preparative HPLC with 40% methanol to give compounds 13 (1.6 mg) and 14 (1.1 mg). Fr 4-3-3-3 was further purified by semi-preparative HPLC with 45% methanol to give compound 5 (1.7 mg).

[0152] Fr 3 (61.15 g) was separated on an MCI column and eluted with a methanol-water solvent system gradient (50% methanol, 60% methanol, 70% methanol, 80% methanol, 90% methanol); the 70% methanol elution portion was further purified by preparative high performance liquid chromatography with 39% methanol to give compound 17 (1.3 mg).

[0153] The monomers of compounds 1-17 obtained in the above steps were mixed to obtain Callicarpa nudiflora extract A.

[0154] Example 2

[0155] The preparation method was the same as that in Example 1 to obtain 3.4 mg of compound 7, 1.8 mg of compound 8, and 2.1 mg of compound 14. The obtained compounds 7, 8, and 14 were mixed to obtain Callicarpa nudiflora extract B.

[0156] Example 3 Structure Identification

[0157] Compound 1: colorless oil, molecular formula C 19 H 30O7Na, ESI-MSm / z: 393.1[M+Na]+. 1H-NMR(600MHz,CD3OD)δ:5.89(1H,br s,H-4),5.79(1H,dd,J=15.4,6.5Hz,H-8),5.65(1H,dd,J=15.4,9.2Hz,H-9),4.41(1H,t ,J=6.5Hz,H-1'),4.36(1H,d,J=7.8Hz,H-7),3.83(1H,m,H-6'),3.67(1H,dd,J=11.9,5. 4Hz,H-6'),3.36(1H,m,H-5'),3.29(m,6H,H-4'),3.22(1H,m,H-3'),3.18(1H,m,H-2'), 2.69(1H,d,J=9.3Hz,H-6),2.44(1H,d,J=16.8Hz,H-2),2.06(1H,d,J=16.8Hz,H-2),1.95 (3H,s,H-11),1.30(3H,d,J=4.8Hz,H-10),1.04(3H,s,H-11),1.01(3H,s,H-12); 13C-NM R(150MHz,CD3OD)δ:37.1(C-1),48.3(C-2),202.1(C-3),126.1(C-4),165.9(C-5),56.8 (C-6),77.0(C-7),138.2(C-8),128.8(C-9),21.0(C-10),23.8(C-11),28.0(C-12),27. 6(C-13),102.5(C-1'),75.3(C-2'),78.0(C-3'),71.5(C-4'),78.1(C-5'),62.7(C-6'). The above data are consistent with those reported in the literature (Zhu LH, Bao TH, Deng Y, et al. Constituents from Apium graveolens and their anti-inflammatory effects[J]. J Asian Nat Prod Res, 2017, 19: 1079-1086), so the compound was identified as (6S,7R)-3-oxo-megastigma-4,8-dien-7-O-β-D-glucoside.

[0158] Compound 2: amorphous powder, molecular formula C 19 H 32O7Na, ESI-MSm / z: 395.2[M+Na]+. 1H-NMR (600MHz, CD3OD) δ: 4.37 (1H, d, J = 7.8Hz, H-1'), 3.92 (1H, m, H-9), 3.87 (1H, dd, J = 5 .1,2.4Hz,H-6'),3.67(1H,dd,J=11.8,5.3Hz,H-6'),3.36(1H,d,J=8.7Hz,H-3'),3.34(1 H,d,J=1.3Hz,H-4'),3.28(1H,m,H-5'),3.19(1H,dd,J=4.3,1.5Hz,H-2'),2.55(1H,m,H- 7),2.45(1H,m,H-3),2.32(1H,td,J=12.1,5.9Hz,H-7),1.83(1H,m,H-2),1.77(3H,s,H-1 3),1.68(1H,m,H-8),1.31(1H,d,J=6.3Hz,H-10),1.21(3H,s,H-11),1.21(3H,s,H-12);1 3C-NMR(150MHz,CD3OD)δ:37.7(C-1),38.4(C-2),35.1(C-3),201.6(C-4),131.7(C-5),1 68.5(C-6),27.1(C-7),36.4(C-8),77.7(C-9),21.8(C-10),27.2(C-11),27.6(C-12),11.8(C-13),104.1(C-1'),75.4(C-2'),77.9(C-3'),71.7(C-4'),78.3(C-5'),62.8(C-6'). The above data are consistent with those reported in the literature (Nguyen TVT, Tran TM, Nguyen TL, et al. Megastigmane Glycosides From Phoebe tavoyana[J]. Natural Product Communications, 2019, 14(6), 1-4), so the compound was identified as phoebenoside A.

[0159] Compound 3: white powder, molecular formula C 19 H 30O7Na, FAB-MSm / z:393.2[M+Na]+. 1H-NMR(600MHz,CD3OD)δ:5.89(1H,br s,H-4),5.79(1H,dd,J=15.4,6.5Hz,H-8),5.66(1H,dd,J=15.4,9.3Hz,H-7),4.41(1H,m,H-9) ,4.36(1H,d,J=7.8Hz,H-1'), 3.83(1H,dd,J=11.8,2.4Hz,H-6'), 3.67(1H,dd,J=11.7,5.4Hz, H-6'),3.37(1H,m,H-3'),3.34(1H,d,J=8.9Hz,H-4'),3.30(1H,d,J=9.2Hz,H-2'),3.18(1H,m ,H-5'),2.69(1H,d,J=9.3Hz,H-6),2.44(1H,d,J=16.8Hz,H-2),2.06(1H,d,J=16.8Hz,H-2),1 .95(3H,d,J=1.3Hz,CH3-13),1.30(3H,d,J=6.4Hz,CH3-10),1.04(3H,s,CH3-12),1.02(3H,s, CH3-11); 13C-NMR (150MHz, CD3OD) δ: 37.1 (C-1), 48.3 (C-2), 202.1 (C-3), 126.1 (C-4), 165.9 ( C-5),56.8(C-6),128.8(C-7),138.2(C-8),77.0(C-9),21.0(C-10),27.6(C-11),28.0(C-12) ,23.8(C-13),102.5(C-1′),75.3(C-2′),78.1(C-3′),71.5(C-4′),78.0(C-5′),62.7(C-6′). The above data are consistent with those reported in the literature (Kuang HX, Yang BY, Xia YG, et al. Chemical constituents from the flower of Datura metel L[J]. Arch Pharm Res, 2008, 31: 1094-7.), so the compound was identified as (6R,9R)-3-oxo-α-ionol-9-O-β-D-glucopyranoside.

[0160] Compound 4: reddish brown oily solid, molecular formula C 19 H 31O7, FAB-MSm / z:371.2[M-H]-. 1H-NMR(600MHz,CD3OD)δ:5.81(1H,s,H-4),4.33(1H,d,J=7.8Hz,H-1'),3.85(1H ,d,J=1.6Hz,H-6'),3.83(1H,m,H-9),3.66(1H,m,H-6'),2.50(1H,d,J=17.4Hz,H -2),2.06(3H,d,J=1.4Hz,H-13),2.00(1H,m,H-6),1.98(1H,d,J=5.8Hz,H-2),1. 81(1H,m,H-7),1.70(1H,m,H-8),1.68(1H,m,H-7),1.62(1H,m,H-8),1.26(3H,d, J=6.3Hz,H-10),1.11(3H,s,H-12),1.03(3H,s,H-11);13C-NMR(150MHz,CD3OD)δ :37.4(C-1),48.1(C-2),202.4(C-3),125.4(C-4),169.9(C-5),52.6(C-6),26.7 (C-7),37.4(C-8),77.9(C-9),21.9(C-10),29.0(C-11),27.5(C-12),25.0(C-13 ), 104.0(C-1′), 75.3(C-2′), 78.2(C-3′), 71.7(C-4′), 77.6(C-5′), 62.8(C-6′). The above spectral data were consistent with those reported in the literature (Matsunami K, Otsuka H, ​​Takeda Y. Structural revisions of blumenol C glucoside and byzantionoside B[J]. ChemPharm Bull (Tokyo), 2010, 58: 438-41.), so the compound was identified as blumenol C glucoside.

[0161] Compound 5: white powder, molecular formula is C 29 H 37 O 15,ESI-MSm / z:625.2[M+H]+. 1H-NMR (600MHz, CD3OD) δ: 1.11 (3H, d, J = 6.2 Hz, Rha-H-6″′), 2.81 (2H, m, H-7), 3.31~4.07 (12H, m, sugar- H,H-8),4.40(1H,d,J=7.9Hz,Glc-H-1″),5.21(1H,s,Rha-H-1″′),6.29(1H,d,J=15.9Hz,H-α),6.58(1H,d d,J=2.1,8.0Hz,H-6),6.69(1H,d,J=8.0Hz,H-5),6.72(1H,d,J=2.0Hz,H-2),6.80(1H,d,J=8.2Hz,H-5′), 6.97(1H,dd,J=2.1,8.2Hz,H-6′),7.07(1H,d,J=2.1Hz,H-2′),7.61(1H,d,J=15.6Hz,H-β); 13C-NMR(150M Hz, CD3OD)δ:131.5(C-1),117.1(C-2),146.8(C-3),146.1(C-4),116.5(C-5),121.3(C-6),36.6(C-7),72 .3(C-8),127.7(C-1′),114.7(C-2′),146.8(C-3′),149.8(C-4′),116.5(C-5′),123.2(C-6′),115.2(C-α The above data are consistent with those reported in the literature (Su BN, Ma LP, Jia Z J. Iridoid and phenylpropanoid glycosides from Pedicularis arteselaeri [J]. Planta Med, 1998, 64 (8): 720-723.), so the compound was identified as isocarpus glycoside.

[0162] Compound 6: amorphous powder, molecular formula C 34 H 43 O 19; FAB-MSm / z: 755.2 [M-H]-. 1H-NMR (600 MHz, CD3OD) δ: 7.60 (1H, d, J = 15.9 Hz, H-β'), 7.08 (1H, d, J = 2.1 Hz, H-2''), 6.99 (1H, dd, J = 8.2, 2.1 Hz, H-6''), 6.82 (1H, d, J = 8.2 Hz, H-5''), 6.71 (1H, d, J = 2.1 Hz, H-2), 6.69 (1H, d, J = 8.0 Hz, H-5), 6.57 (1H, dd, J = 8.1, 2.1 Hz, H-6), 6.26 (1H, d, J = 15.9 Hz, H-α'), 5.29 (1H, d, J = 1.8 Hz, H-1''), 5.22 (1H, d, J = 2.7 Hz, H-1'''), 4.94 (1H, t, J = 9.6 Hz, H-4'), 4.39 (1H, d, J = 7.9 Hz, H-1'), 4.07 (1H, m, H-α), 3.88 (1H, m, H-3'), 3.85 (1H, d, J = 9.2 Hz, H-4'''), 3.74 (1H, m, H-α), 3.68 (1H, dd, J = 9.4, 3.3 Hz, H-3''), 3.63 (1H, s, H-5'''), 3.56 (1H, m, H-5'), 3.42 (1H, m, H-4''), 2.80 (2H, m, H-β), 1.13 (3H, d, J = 6.2 Hz, H-6''); 13C-NMR (150 MHz, CD3OD) δ: 131.5 (C-1), 116.2 (C-2), 146.1 (C-3), 144.6 (C-4), 117.0 (C-5), 121.2 (C-6), 72.2 (C-α), 36.5 (C-β), 104.1 (C-1′), 75.9 (C-2′), 80.4 (C-3′), 70.3 (C-4′), 76.4 (C-5′), 62.2 (C-6′), 102.1 (C-1″), 72.4 (C-2″), 80.1 (C-3″), 72.5 (C-4″), 68.8 (C-5″), 18.7 (C-6″), 111.4 (C-1″′), 78.5 (C-2″′), 80.0 (C-3″′), 74.8 (C-4″′), 65.7 (C-5″′), 127.6 (C-1″″), 115.2 (C-2″″), 146.9 (C-3″″), 149.9 (C-4″″), 116.6 (C-5″″), 123.3 (C-6″″), 114.6 (C-α'), 148.0 (C-β'), 168.2 (C=O). The spectral data is consistent with the literature reports ( Bedir E, Kirmizibekmez H, et al. Secondary metabolites from Phlomisoppositiflora[J]. Nat Prod Res, 2005, 19: 493-501.), thus identifying the compound as myricoside.

[0163] Compound 7: white powder; molecular formula C 31 H 40 O 15Na; ESI-MSm / z:675.2[M+Na]+. 1H-NMR (500MHz, CD3OD)δ:7.67(1H,d,J=15.9Hz,H-α'),7.20(1H,d,J=1.9Hz,H-2'),7.09(1H,dd,J=8.2,2.0Hz,H-6'),6.83(1H ,d,J=7.8Hz,H-5'),6.82(1H,d,J=2.4Hz,H-2),6.74(1H,d,J=8.1Hz,H-5),6.69(1H,dd,J=8.2,2.1Hz,H-6),6.38(1H,d,J=15.9H z,H-β'),5.21(1H,s,H-1),4.93(1H,t,J=9.3Hz,H-4″),4.39(1H,d,J=7.9Hz,H-1″),4.07(1H,t,J=9.7Hz,H-3″),3.89(3H,s,OC H3),3.85(1H,m,H-3),3.82(3H,s,OCH3),2.83(2H,m,H-α),1.11(3H,d,J=6.2Hz,H-6); 13C-NMR(150MHz,CD3OD)δ:132.9(C-1),1 12.8(C-2),147.9(C-3),147.5(C-4),117.1(C-5),121.1(C-6),36.5(C-7),72.3(C-8),127.6(C-1'),111.8(C-2'),147.4(C-3 '),149.4(C-4'),116.5(C-5'),124.4(C-6'),149.4(C-7'),115.1(C-8'),168.3(C-9'),104.2(C-1″),76.0(C-2″),81.5(C-3″) ,70.4(C-4″),76.0(C-5″),62.4(C-6″),103.0(C-1″′),72.1(C-2″′),72.3(C-3″′),73.8(C-4″′),70.6(C-5″′),18.4(C-6″′),56.4(3'-OCH3),56.5(3-OCH3). The above data are consistent with those reported in the literature (Chen Yi, Ye Caiyun, Zhao Yong. Study on the chemical components of phenylethanol in Radix Saposhnikoviae Radix [J]. Chinese Herbal Medicine, 2017, 48(19):3941-3944.), so the compound was identified as cistancheside D.

[0164] Compound 8: brown-yellow oil; molecular formula C 31 H 40 O 15Na; ESI-MS: m / z: 675.2[M+Na]+. 1H-NMR (CD3OD, 600MHz) δ: 7.67 (1H, d, J = 15.9Hz, H-7"'), 7.21 (1H, d, J = 2.0Hz, H-2"'), 7.09 (1H, dd, J = 8.2, 2.0Hz ,H-6"'),6.82(2H,dd,J=8.2,10.0Hz,H-5,5"'),6.75(1H,d,J=2.1Hz,H-2),6.70(1H,dd,J=8.2,2.2Hz,H-6),6.38 (1H,d,J=15.9Hz,H-8"'),5.21(1H,brs,H-1"),4.93(1H,t,J=9.4Hz,H-4'),4.39(1H,d,J=7.9Hz,H-1'),3.90(3H ,s,OCH3),3.82(3H,s,OCH3),2.83(2H,m,H-8),1.11(3H,d,J=6.2Hz,H-6”); 13C-NMR(CD3OD,150MHz)δ:168.3(C-9 ”'),151.1(C-4”’),149.5(C-3”’),147.9(C-7”’),147.6(C-3),147.4(C-4),132.9(C-1),127.5(C-1”’),124.4( C-6"'),121.1(C-6),117.1(C-5),116.5(C-5"'),115.0(C-8"'),112.8(C-2),111.7(C-2"'),104.2(C-1'),103.0 (C-1”), 81.5 (C-3’), 76.2 (C-2’), 76.1 (C-5’), 73.8 (C-4”), 72.4 (C-2”), 72.1 (C-7), 72.1 (C-3”), 70.6 (C-4’), 70.4 (C-5”), 62.4 (C-6’), 56.5 (4-OCH3), 56.4 (4”’-OCH3), 36.6 (C-8), 18.4 (C-6”). The above data are consistent with those reported in the literature (Teborg D, Junior P. Martynoside and the novel dimeric openchain monoterpene glucoside digipen-stroside from Penstemondigitalis [J]. Planta Med, 1989, 55: 474-476.), so the compound was identified as rehmannia glutinin.

[0165] Compound 9: brown oil; molecular formula C 23 H25 O 11 ;ESI-MSm / z 477[MH]-. 1H-NMR (CD3OD, 600MHz) δ: 7.58 (1H, d, J = 2.3Hz, H-7″), 7.04 (1H, d, J = 2.3Hz, H-2″), 6.90 (1 H,dd,J=8.2,2.3Hz,H-6″),6.79(1H,d,J=8.2Hz,H-5″),6.68(1H,d,J=2.3Hz,H-2),6.65(1 H,d,J=8.1Hz,H-5),6.64(1H,d,J=15.4Hz,H-8″),6.55(1H,dd,J=8.0,2.3Hz,H-6),4.51(1 H,dd,J=2.4,12.0Hz,H-6'a),4.35(m,1H),4.33(1H,t,H-6'b),3.96(1H,m,H-α),3.72(1H,m ,H-α),2.80(2H,m,H-β);13C-NMR(CD3OD,150MHz)δ:131.4(C-1),116.5(C-2),146.8(C-3) ,146.1(C-4),116.3(C-5),121.2(C-6),72.4(C-α),36.7(C-β),104.6(C-1'),75.1(C-2') ,77.9(C-3'),71.7(C-4'),75.5(C-5'),64.6(C-6'),127.7(C-1″),114.8(C-2″),147.2(C-3″),149.6(C-4″),117.1(C-5″),123.2(C-6″),144.7(C-7″),115.0(C-8″),169.2(C-9″). The above data are basically consistent with the data reported in the literature ( T, D, Phenylethanoid glycosides from Scutellariagalericulata[J].Turk J Chem, 2002, 26(4):465-471.), and thus the compound was identified as calceolarioside B.

[0166] Compound 10: white powder, molecular formula C 26 H 31 O 11, FAB-MSm / z:519.2[M-H]-. 1H-NMR(600MHz,CD3OD)δ:7.15(1H,dd,J=8.3,1.5Hz,H-5),7.04(1H,d,J=2.0Hz,H-2),6.95(1H,d,J=1.9Hz,H-2′),6.93(1H,d d,J=8.4,2.0Hz,H-6),6.82(1H,dd,J=8.1,2.0Hz,H-6′),6.77(1H,d,J=8.1Hz,H-5′),4.72(1H,d,J=4.4Hz,H-1″),4.24-4.275 (2H,m,H-9,H-9′),3.88(3H,s,OCH3),3.86(1H,s,OCH3),3.85(s,0H),3.69(2H,m,H-9′),3.49(1H,m,H-6″a),3.47(1H,m,H-6″ b),3.39-3.40(4H,m,H-2″-5″),3.39(d,J=1.3Hz,0H),3.14(2H,m,H-8,H-8′);13C-NMR(150MHz,CD3OD)δ:137.5(C-1),111.6( C-2),147.5(C-3),151.0(C-4),118.0(C-5),119.8(C-6),87.1(C-7),55.5(C-8),72.7(C-9),133.7(C-1′),111.0(C-2′),147 .4(C-3′),149.1(C-4′),116.1(C-5′),120.1(C-6′),87.5(C-7′),55.4(C-8′),72.7(C-9′),56.4(4-OCH3),56.7(4'-OCH3),1 02.9(C-1″),74.9(C-2″),77.8(C-3″),71.3(C-4″),78.2(C-5″),62.5(C-6″). The above data are consistent with those reported in the literature (Li Ning, Tan Ninghua, Zhou Jun. A new lignan glycoside from Curculigo orchioides[J]. Yunnan Plant Research, 2003(06):711-716.), so the compound was identified as 4,4'-dimethoxy-3'-hydroxy-7',9-diepoxylignan-3-O-β-D-glucopyranoside.

[0167] Compound 11 is a pale yellow powder with the molecular formula C 21 H 17 O 12 ,ESI-MSm / z:462.1[MH] - . 1H-NMR(600MHz,CD3OD)δ:7.45(1H,dd,J=8.4,2.1Hz,H-6′),7.43(1H,d,J=2.2Hz,H-2′),6.94(1H,d,J=8.3Hz,H-5′),6.79(1 H,d,J=2.2Hz,H-8),6.64(1H,s,H-3),6.51(1H,d,J=2.2Hz,H-6),5.21(1H,d,J=7.9Hz,H-1″),3.56-4.19(4H,m,H-2″-H-5″); 13 C-NMR(150MHz,CD3OD)δ:166.9(C-2),104.2(C-3),184.1(C-4),163.0(C-5), 101.4(C-6),164.4(C-7),96.0(C-8),158.9(C-9),107.3(C-10),123.5(C-1′ ),114.3(C-2′),147.1(C-3′),151.2(C-4′),116.8(C-5′),120.5(C-6′),101 .0(C-1″),74.4(C-2″),77.0(C-3″),72.8(C-4″),76.7(C-5″),170.8(C-6″). The above data are consistent with those reported in the literature (Rabelo AS, Oliveira ID, AG et al. Antinociceptive, anti-inflammatory and antioxidant activities of aqueous extract from Remirea maritima (Cyperaceae). [J]. J Ethnopharmacol, 2013, 145: 11-7.), and thus the compound was identified as luteolin-7-O-glucuronide.

[0168] Compound 12: Pale yellow powder, molecular formula C 21 H 19 O 11,ESI-MSm / z:447[M-H]-. 1H-NMR(600MHz,CD3OD)δ:12.99(1H,s,5-OH),7.47(1H,dd,J=8.5,2.3Hz,H-6'),7.4 5(1H,d,J=2.2Hz,H-2'),7.33(1H,d,J=8.5Hz,H-5'),6.62(1H,s,H-3),6.46(1H,d,J =2.1Hz,H-8),6.22(1H,d,J=2.1Hz,H-6),4.95(1H,d,J=7.6Hz,H-1″),3.93-3.44(6H ,m,sugar-H);13C-NMR(150MHz,CD3OD)δ:165.5(C-2),103.2(C-3),183.8(C-4),159. 5(C-5),100.3(C-6),166.3(C-7),95.1(C-8),163.3(C-9),105.4(C-10),119.8(C-1 '),114.9(C-2'),148.7(C-3'),150.0(C-4'),118.0(C-5'),127.3(C-6'),105.1(C- 1″), 71.3(C-2″), 74.8(C-3″), 78.5(C-4″), 77.5(C-5″), 62.4(C-6″). The above data are consistent with those reported in the literature (Ling Yun, He Banzuo, Bao Yanyan, et al. Study on the chemical components of duckweed [J]. Chinese Herbal Medicine, 1999(2):88-90.), so the compound was identified as luteolin-7-O-β-D-glucoside.

[0169] Compound 13: Pale yellow powder, molecular formula C 21 H 19 O 11,ESI-MSm / z:447[M-H]-. 1H-NMR(600MHz,CD3OD)δ:12.92(1H,s,5-OH),7.47(1H,d,J=8.5Hz,H-6'),7.45(1H,brs ,H-2'),7.33(1H,d,J=8.5Hz,H-5'),6.62(1H,s,H-3),6.46(1H,d,J=2.1Hz,H-8),6.22(1 H,d,J=2.1Hz,H-6),4.95(1H,d,J=7.6Hz,H-1″),3.93-3.44(6H,m,H-2″-6″); 13C-NMR(1 50MHz, CD3OD)δ:183.8(C-4),166.3(C-2),165.5(C-7),163.3(C-5),159.5(C-9),150.0( C-4'),148.7(C-3'),127.3(C-1'),119.8(C-6'),118.0(C-5'),114.9(C-2'),105.4(C- 10),105.1(C-3),103.2(C-1″),100.3(C-6),95.1(C-8),78.5(C-3″),77.5(C-5″),74.8( C-2″), 71.3(C-4″), 62.4(C-6″). The above data are consistent with the literature report (Ji Min, Li Shujuan, Ma Chaomei. Study on the chemical components of the inflorescence of narrow-leaved blue pot flower and its antioxidant and α-glucosidase inhibition activities [J]. Journal of Inner Mongolia University (Natural Science Edition), 2014, 45(4): 398-403.), so the compound was identified as luteolin-4'-O-β-D-glucoside.

[0170] Compound 14: Pale yellow powder, molecular formula C 21 H 19 O 10,ESI-MSm / z:447[M-H]-. 1H-NMR(600MHz,CD3OD)δ:7.90(2H,d,J=8.4Hz,H-6',H-2'),6.94(2H,d,J=8.4Hz,H-5',H-3'),6.84(1H,s,H-3),6.67(1H,s,H-8),6.51(1H,d, J=2.0Hz, H-6), 5.08 (1H, d, J=6.3Hz, H-1″); 13C-NMR (150MHz, CD3OD) δ: 166.8 (C-2), 104.1 (C-3), 184.1 (C-4), 159.0 (C-5), 101.2 (C-6), 164.8 (C-7), 96.1(C-8), 162.9(C-9), 107.1(C-10), 123.0(C-1'), 129.7(C-2',6'), 117.1(C-3',5'), 163.2(C-4'), 101.6(C-1″), 74.7(C-2″), 77.9(C-3″), 71.3(C-4″), 78.4(C-5″), 62.5(C-6″). The above data are consistent with those reported in the literature (Ren Dongchun, Yang Nianyun, Qian Shihui, et al. Study on the chemical constituents of the aerial parts of Chuanxiong [J]. China Journal of Traditional Chinese Medicine, 2007, 2(4): 1418.), so the compound was identified as cosmosin.

[0171] Compound 15: Pale yellow powder, molecular formula C 23 H 21 O 12,ESI-MSm / z:489[M-H]-. 1H-NMR(600MHz,CD3OD)δ:7.43(1H,dd,J=8.3,2.3Hz,H-6'),7.41(1H,d,J=2.2Hz,H-2'),6.91(1H, d,J=8.3Hz,H-5'),6.77(1H,d,J=2.2Hz,H-8),6.61(1H,s,H-3),6.47(1H,d,J=2.2Hz,H-6),5.30(1H d, J=1.8Hz, H-1"'), 5.21 (1H, d, J=7.7Hz, H-1"), 1.34 (3H, d, J=6.2Hz, H-6"'); 13C-NMR (150MHz, CD3OD) δ: 184.0 (C-4), 166. 9(C-2),164,4(C-7),163,0(C-5),159,1(C-9),151,3(C-4′),147,1(C-3′),123,5(C-1′),120,5(C-6′),116,8(C-5′),114,3 (C-2′),107,1(C-10),104,2(C-3),102,6(C-1″′),100,9(C-6),99,8(C-1″),95,9(C-8),79,1(C-5″),79,0(C-3″),78,3(C-2″),74,0(C-4″′),72,2(C-3″′),72,2(C-2″′),71,4(C-4″),70,0(C-5″′),62,4(C-6″),18,3(C-6″′). The above data are consistent with those reported in the literature (Raimundo RG,Nascimento,Jackelyne A,et al.New flavonoids from Margaritopsiscarrascoanawith antioxidant activity[J].Química Nova,2015,38(1),60-65.),so the compound was identified as luteolin-7-O-β-L-rhamnopyranosyl(1,2)-β-D-glucopyranoside.

[0172] Compound 16: yellow powder, molecular formula C 27 H 29 O 14,ESI-MSm / z:577.2[M-H]-. 1H-NMR (CD3OD, 600MHz) δ: 7.78 (2H, d, H-2′, 6′), 6.94 (2H, m, H-3′, 5′), 6.79 (1H, d, J = 2.2H z,H-8),6.66(1H,s,H-3),6.46(1H,d,J=2.2Hz,H-6),5.30(1H,d,J=1.8Hz,H-1″′),5.21(1H ,d,J=7.6Hz,H-1″),1.34(3H,d,J=6.2Hz,H-6″′); 13C-NMR(CD3OD,150MHz)δ:184.0(C-4),1 66.8(C-7),164.4(C-2),163.0(C-5),162.9(C-4′),159.0(C-9),129.6(C-2′,6′),123.0(C -1′),117.1(C-3′,5′),107.0(C-10),104.1(C-3),102.5(C-6),101.0(C-1″′),99.8(C-1″ ),95.9(C-8),79.1(C-2″),79.0(C-3″),78.3(C-5″),74.0(C-4″′),72.2(C-2″′),72.2(C-3 ″′), 71.4(C-4″), 70.0(C-5″′), 62.4(C-6″), 18.3(C-6″′). The above data are basically consistent with those reported in the literature (Chen Lin, Xiang Caipeng, Han Jiaxin, et al. Study on the chemical composition and anticholinesterase activity of pepper in South China [J]. Natural Product Research and Development, 2018, 30(9): 1569.), so the compound was identified as apigenin-7-O-β-D-neohesperidin.

[0173] Compound 17: yellow powder, molecular formula C 20 H 29O4, ESI-MSm / z:333.2[M-H]-. 1H-NMR(CD3OD,600MHz)δ:7.73(2H,dd,J=5.7,3.3Hz,H-3,6),7.63(2H,dd,J=5.8,3.3Hz,H-4,5),4.30(4H,m,H-1'),4.2 3(2H,m,H-1”),1.72(3H,m,H-2’,2”),1.45(4H,m,H-3’,a”),1.36(6H,m,H-3”,4”,5”),0.95(9H,m,3×CH3,H-4’,6”,b”); 13C-NMR(CD3OD,150MHz)δ:169.3(CO),133.6(C-4),132.4(C-5),130.6(C-1,2),129.9(C-3,6),69.1(C-1”),66.7(C-1' ),40.2(C-2”),31.7(C-2’),31.6(C-3”),30.1(C-4”),25.0(Ca”),24.0(C-5”),20.3(C-3’),14.4(C-4’,6”),11.4(Cb”). The above hydrogen and carbon spectrum data were consistent with the literature (Gao Chenghai, Yi Xiangqian, Lin Lin, et al. Study on the chemical composition and structure-activity relationship of marine phthalates [J]. Natural Product Research and Development, 2013, 25(10): 1320-1324.), so the compound was identified as 2-O-butyl-1-O-(2'-ethylhexyl)benzene-1,8-dicarboxylate.

[0174] Purity identification:

[0175]

[0176]

[0177] Example 3 Evaluation of anti-inflammatory activity

[0178] 3.1 Cell culture and toxicity evaluation

[0179] BV2 microglial cells were cultured in DMEM containing 10% fetal bovine serum at 37°C and 5% CO2, and passaged when the cells reached 90% viability. The CCK8 assay was used to determine the effects of the compounds on BV2 cell viability. Meanwhile, BV2 cells in the logarithmic growth phase were seeded in 96-well plates at a density of 9 × 10 cells per well. 3After cells adhered, the cells were divided into a blank group, a dexamethasone group, and a drug-treated group (25 μmol / L). Each group had three replicate wells. The cells were cultured at 37°C and 5% CO2 for 24 hours. 10% CCK-8 medium was added and the cells were incubated at 37°C for 2 hours. The absorbance was then measured at 450 nm.

[0180] 3.2 Determination of NO content

[0181] BV2 cells were seeded in 96-well plates at a density of 9 × 10 3 After the cells adhered, the experiment was divided into a blank group, an LPS group, a dexamethasone group, and groups treated with different concentrations of dexamethasone, with three replicates per group. Fresh culture medium was added to the blank group, while the dexamethasone and dexamethasone groups were pretreated for 3.5 hours before LPS stimulation. Then, along with the LPS group, dexamethasone at a final concentration of 1 μg / mL LPS was added. After 24 hours of culture, the supernatant was collected and assayed strictly according to the instructions of the NO detection kit.

[0182] 3.3 Statistical analysis

[0183] GraphPad Prism 8.0 software was used for data processing and statistical analysis. The NO inhibition rate was used to represent the anti-inflammatory activity of the compounds in vitro. Each experiment was repeated 3 times, and all values ​​are expressed as mean ± SD.

[0184] 3.4 Results Statistics

[0185] Macrophages play an important role in the occurrence and development of inflammation. When they are invaded by exogenous bodies, they will secrete certain pro-inflammatory and anti-inflammatory factors, which play an important role in the body's inflammatory response. Therefore, inhibiting the release of inflammatory mediators and the activation of related signaling pathways is an important means to relieve inflammation. NO is a noteworthy inflammatory mediator. Excessive NO in the central nervous system will activate microglia and cause inflammatory responses, and it has neurotoxicity. It is closely related to the occurrence and development of various inflammatory diseases. It is generally considered to be a sign of inflammation and used as an in vitro anti-inflammatory drug screening model. We used LPS to stimulate BV2 cells to produce the inflammatory mediator NO to conduct a preliminary screening of the anti-inflammatory activity of the isolated compounds. First, the CCK8 method was used to determine the effect of the compound on the survival rate of BV2 cells to ensure that subsequent experiments were carried out under non-toxic conditions. The experimental results showed that when the concentration of the compound was 25μmol·L -1 When the BV2 cell viability was higher than 80%, all compounds were tested. We then used the Griess method to further test the inhibitory effect of different extracts on LPS-induced NO production in BV2 cells. The results are as follows:

[0186]

[0187] From the data in the table above, it can be seen that compounds 1-17 all exhibited certain anti-inflammatory effects, among which compound 7 had a significantly better anti-inflammatory effect than other compounds and dexamethasone. At the same time, the anti-inflammatory effect of Callicarpa nudiflora extract A (a mixture of compounds 1-17) reached 50.97±0.04 (25μmol·L -1 The anti-inflammatory effect of Callicarpa nudiflora extract A (a mixture of compounds 7, 8, and 14) reached 48.64±0.02 (25 μmol·L -1 )(%), which is superior to any of the monomeric compounds, proving that the combination achieves a synergistic anti-inflammatory effect, and the extract has clear ingredients, which has obvious advantages over the Callicarpa nudiflora extract in the prior art.

[0188] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Callicarpa nudiflora extract, characterized in that Including (6S,7R)-3-oxo-megastigma-4,8-dien-7-O-β-D-glucoside, phoebenoside A, (6R,9R)-3-oxo-α-ionol-9-O-β-D-glucopyranoside, blumenol C glucoside, isovanoscoside, myricoside, cistanchoside D, rutin, calceolarioside B, 4,4'-dimethoxy-3'-hydroxy-7',9-diepoxylignan-3-O-β-D-glucopyranoside, luteolin-7-O-glucuronide, luteolin-7-O-β-D-glucoside, luteolin-4'-O-β-D-glucoside, cosmosin, luteolin-7-O-β-L-rhamnopyranosyl(1,2)-β-D-glucopyranoside, apiol-7-O-β-D-neohesperidin, and 2-O-butyl-1-O-(2'-ethylhexyl)benzene-1,8-dicarboxylate.

2. The Callicarpa nudiflora extract according to claim 1, characterized in that The extract of Callicarpa nudiflora contains (6S,7R)-3-oxo-megastigma-4,8-dien-7-O-β-D-glucoside, phoebenoside A, (6R,9R)-3-oxo-α-ionol-9-O-β-D-glucopyranoside, blumenol C glucoside, isocarpus glycoside, myricoside, cistancheside D, rehmannia glutinosa, calceolariosideB, 4,4'-dimethoxy-3'-hydroxy-7',9-diepoxylignan-3-O-β-D-glucopyranoside, luteolin-7-O-glucuronide, luteolin-4'-O-β-D-glucoside, luteolin-7-O- The mass ratio of β-L-rhamnopyranosyl(1,2)-β-D-glucopyranoside, apigenin-7-O-β-D-neohesperidin and 2-O-butyl-1-O-(2'-ethylhexyl)benzene-1,8-dicarboxylate is 1-5:5-15:1-5:2-6:0.5-5:10-15:1-10:0.1-1.5:1-5:3-8:0.5-4:3-8:0.5-3:0.5-3:1-3:1-5:0.5-4.

3. The method for preparing the Callicarpa nudiflora extract according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1, cold soaking of Callicarpa nudiflora leaves in water, followed by heating and decocting, combining the filtrates, and concentrating under reduced pressure to obtain extract 1, which was suspended in water, extracted with ethyl acetate, and the solvent was recovered under reduced pressure to obtain extract 2; S2 and extract 2 were mixed with silica gel and crudely separated by silica gel column chromatography. Gradient elution was performed with dichloromethane-methanol 600:1 to 1:

1. The three components with the largest extraction amount were selected, and the order of polarity from high to low was Fr 6, Fr 4, and Fr 3; S3 and Fr 6 were separated on an MCI column and eluted with a methanol-water solvent system with a gradient of 30 to 90% methanol. The two components with the largest extraction amounts were selected, and in descending order of polarity, they were Fr 6-1-4 and Fr 6-1-3; Among them, Fr 6-1 was subjected to silica gel column chromatography with a gradient elution of dichloromethane-methanol 30:1 to 1:1, and the two components with the largest extraction amount were selected, in descending order of polarity, as Fr 6-1-4 and Fr 6-1-3; Fr 6-1-3 was subjected to ODS column chromatography with isocratic elution of methanol-water at a ratio of 45-50:50-55, and isocratic separation using 42-48% methanol and 40-45% methanol, respectively, to give compounds 8 and 11; Fr 6-1-4 was further separated by preparative liquid chromatography using 43-46% methanol and 45-50% methanol, respectively, to give compounds 15 and 16; Fr 6-4 was separated by normal phase silica gel column chromatography, and the two components with the largest extraction amount were selected in descending order of polarity, namely Fr 6-4-2 and Fr 6-4-1; Fr 6-4-1 was separated by preparative liquid phase with 50-55% methanol to obtain compound 1; Fr 6-4-2 was separated by preparative liquid phase with 52-58% methanol to obtain compounds 4 and 7; S4 and Fr 4 were separated by reverse phase YMC column chromatography under pressure using a methanol-water solvent system gradient elution of 60-95% methanol. The three components with the largest extraction amount were selected in descending order of polarity, namely Fr 4-1, Fr 4-2, and Fr 4-3. Among them, Fr 4-1 was separated by normal phase silica gel column chromatography, and dichloromethane-methanol 20:1 to 1:1 gradient elution was used to obtain compounds 2 and 6; component Fr4-2 was separated on a silica gel column, and dichloromethane-methanol 50:1 to 5:1 gradient elution was used; and then the preparative liquid phase was passed through 52-58% methanol to obtain compounds 3 and 10; Component Fr 4-3 was separated by Sephadex LH-20 column and methanol, and the two components with the largest extraction amount were selected, which were Fr 4-3-1 and Fr 4-3-3 in descending order of polarity; Fr 4-3-1 was separated by normal phase silica gel column chromatography with a gradient elution of dichloromethane-methanol from 20:1 to 5:1 to obtain compounds 9 and 12; the elution ratios were dichloromethane:methanol at 10:1 and 7:1; Fr 4-3-3 was separated by normal phase silica gel column chromatography with a gradient elution of dichloromethane-methanol from 30:1 to 1:

1. The two components with the largest extraction amount were selected and ranked in descending order of polarity as Fr 4-3-3-3 and Fr 4-3-3-1. Fr 4-3-3-1 was further purified by preparative liquid phase with 42-48% methanol isocratic elution to give compounds 13 and 14; Fr 4-3-3-3 was further purified by preparative liquid phase with 42-50% methanol to give compound 5; S5 and Fr 3 were separated on an MCI column, eluted with a methanol-water solvent system of 40-95% methanol by gradient elution, and then separated by preparative liquid chromatography with 45-55% methanol to obtain compound 17; Among them, compounds 1-17 are (6S,7R)-3-oxo-megastigma-4,8-dien-7-O-β-D-glucoside, phoebenoside A, (6R,9R)-3-oxo-α-ionol-9-O-β-D-glucopyranoside, blumenol C, respectively. glucoside, isocarpus glycoside, myricoside, cistancheside D, rutin, calceolarioside B, 4,4'-dimethoxy-3'-hydroxy-7',9-diepoxylignan-3-O-β-D-glucopyranoside, luteolin-7-O-glucuronide, luteolin-7-O-β-D-glucoside, luteolin-4'-O-β-D-glucoside, cosmosin, luteolin-7-O-β-L-rhamnopyranosyl(1,2)-β-D-glucopyranoside, apiol-7-O-β-D-neohesperidin, 2-O-butyl-1-O-(2'-ethylhexyl)benzene-1,8-dicarboxylate.

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